Cell-Cycle Stress-Related Proteins and Methods of Use in Plants

ABSTRACT

A transgenic plant transformed by a Cell Cycle Stress-Related Protein (CCSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated CCSRPs, and isolated nucleic acid coding CCSRPs, and vectors and host cells containing the latter.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of allowed U.S. Nonprovisional patentapplication Ser. No. 10/768,511, filed Jan. 30, 2004, which is adivisional of U.S. Nonprovisional patent application Ser. No. 09/828,062filed Apr. 6, 2001, now U.S. Pat. No. 6,710,229, and claims the prioritybenefit of U.S. Provisional Application Ser. No. 60/196,001 filed Apr.7, 2000, each of which is hereby incorporated in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingproteins that are associated with abiotic stress responses and abioticstress tolerance in plants. In particular, this invention relates tonucleic acid sequences encoding proteins that confer drought, cold,and/or salt tolerance to plants.

2. Background Art

Abiotic environmental stresses, such as drought stress, salinity stress,heat stress, and cold stress, are major limiting factors of plant growthand productivity. Crop losses and crop yield losses of major crops suchas rice, maize (corn) and wheat caused by these stresses represent asignificant economic and political factor and contribute to foodshortages in many underdeveloped countries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of desiccation. However,if the severity and duration of the drought conditions are too great,the effects on plant development, growth and yield of most crop plantsare profound. Furthermore, most of the crop plants are very susceptibleto higher salt concentrations in the soil. Continuous exposure todrought and high salt causes major alterations in the plant metabolism.These great changes in metabolism ultimately lead to cell death andconsequently yield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold and salttolerance in model, drought-and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

Therefore, what is needed is the identification of the genes andproteins involved in these multi-component processes leading to stresstolerance. Elucidating the function of genes expressed in stresstolerant plants will not only advance our understanding of plantadaptation and tolerance to environmental stresses, but also may provideimportant information for designing new strategies for crop improvement.

One group of proteins that seems to be associated with stress responsesin plants and animals are proteins related to cell division and the cellcycle. Evidence of this relationship is found in the fact that differentenvironmental stresses related to soil water content, incident light andvarying leaf temperature all result in a change in the cell divisionrate. For example, in sunflower leaves, there is a lengthening of thecell cycle associated with stress and aging. Cells in the sunflowerleaves tend to arrest at the G1 phase of cell cycle, with no change inthe duration of the S-G2-M phases of the cell cycle. Similarobservations have been made in other plant species placed underenvironmental stresses such as sucrose starvation (Van't Hof 1973Bookhaven Symp. 25:152), oxidative stress (Reichheld et al. 1999 PlantJ. 17:647), and water depletion (Schuppler et al. 1998 Plant Physiol.117:667). These observations and results suggest that there is animportant “checkpoint” in the regulation of the cell cycle at the G1-Stransition, while other studies suggest another “checkpoint” at the G2-Mtransition with an arrest of the cells at the G2 phase. It has also beendetermined that temperature affects the duration of all cell cyclephases by a similar proportion without the preferential arrest in anyparticular phase of the cell cycle (Tardieu and Granier, 2000 Plant Mol.Biol. 43:555). Although these aforementioned responses to environmentalstresses differ, each of these results demonstrates theinterconnectivity of the stress response system of these plants and thecell division proteins therein.

The prior art describes several proteins associated with cell divisionand the cell cycle. One such protein, or protein complex, is thecyclin-CDK complex, which controls progression of the cell cycle phases.Cyclin-dependent protein kinases (CDKs) require cyclin binding foractivity and are now widely recognized players at the checkpoints of theeukaryotic cell cycle. The widespread importance of CDKs was realizednearly ten years ago from independent genetic approaches in yeast andbiochemical studies of mitotic controls in fertilized eggs of marineinvertebrates.

One known component of a CDK complex is a Cdc2 protein termed p34cdc2,which is required at both control points (G1-S and G2-M). Several otherCdc2 homologs have been isolated from human and plant species includingyeast. One such yeast homolog is Cdc48, which plays a role in thespindle pole body separation in Saccharomyces cerevisiae. Another Cdc2homolog has been described in Arabidopsis (Feiler et al. 1995 EMBO J14:5626) that is highly expressed in the proliferating cells of thevegetative shoot, root, floral inflorescence and flowers and in rapidlygrowing cells. The Arabidopsis Cdc48 gene is up regulated in thedeveloping microspores and ovules and down regulated in mostdifferentiated cell types. In addition, this gene has been localized tothe nucleus and during cytokinesis to the fragmoplast.

Another group of proteins involved in cell division and the cell cycleare pRB proteins. Growing evidence suggests that pRB-like proteins inplants might be among the nuclear targets of plant CDKs. In mammals, thepRB is central to the regulation of the G1-to-S transition.Phosphorylation of mammalian pRB by cyclin D-and cyclin E-dependentkinases renders the pRB inactive and thereby represses the S phase andpromotes DNA replication. Significantly, the pRB-binding motif LXCXE(where X denotes any amino acid) is found in all known plant D cyclins.In plants, LXCXE-dependent interactions between D cyclins fromArabidopsis and maize pRB proteins have been demonstrated in vitro andin a yeast two-hybrid assay. The role of pRBs in plant cell divisionrelated signaling cascades is further supported by the fact that severalpRBs, and in particular, the maize pRB, contain multiple putative CDKphosphorylation sites and are efficiently phosphorylated in vitro bymammalian G1-and S-specific CDKs. Maize pRB proteins are also known toundergo changes in phosphorylation during the transition toendoreduplication in the endosperm. However, phosphorylation by plantCDKs remains to be demonstrated.

Therefore, although several plant cell cycle proteins have beenelucidated, the prior art has yet to describe the plant cell cyclesignal transduction cascades in detail. The prior art also fails todescribe the relation between plant cell cycle proteins and a plant'sresponse to environmental stress such that a stress tolerant plant canbe generated. Accordingly, there is a need to identify genes expressedin stress tolerant plants that have the capacity to confer stressresistance to its host plant and to other plant species. Newly generatedstress tolerant plants will have many advantages, such as increasing therange that crop plants can be cultivated by, for example, decreasing thewater requirements of a plant species.

SUMMARY OF THE INVENTION

This invention fulfills in part the need to identify new, unique cellcycle proteins capable of conferring stress tolerance to plants uponover-expression. The present invention provides a transgenic plant celltransformed by a Cell Cycle Stress-Related Protein (CCSRP) codingnucleic acid, wherein expression of the nucleic acid sequence in theplant cell results in increased tolerance to environmental stress ascompared to a wild type variety of the plant cell. Namely, describedherein are the cell cycle proteins 1) Cell Cycle-1 (CC-1); 2) CellCycle-2 (CC-2) and 3) Cell Cycle-3 (CC-3), all from Physcomitrellapatens.

The invention provides in some embodiments that the CCSRP and codingnucleic acid are that found in members of the genus Physcomitrella. Inanother preferred embodiment, the nucleic acid and protein are from aPhyscomitrella patens. The invention provides that the environmentalstress can be salinity, drought, temperature, metal, chemical,pathogenic and oxidative stresses, or combinations thereof In preferredembodiments, the environmental stress can be drought or coldtemperature.

The invention further provides a seed produced by a transgenic planttransformed by a CCSRP coding nucleic acid, wherein the plant is truebreeding for increased tolerance to environmental stress as compared toa wild type variety of the plant. The invention further provides a seedproduced by a transgenic plant expressing a CCSRP, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant.

The invention further provides an agricultural product produced by anyof the below-described transgenic plants, plant parts or seeds. Theinvention further provides an isolated CCSRP as described below. Theinvention further provides an isolated CCSRP coding nucleic acid,wherein the CCSRP coding nucleic acid codes for a CCSRP as describedbelow.

The invention further provides an isolated recombinant expression vectorcomprising a CCSRP coding nucleic acid as described below, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

The invention further provides a method of producing a transgenic plantwith a CCSRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant comprising: (a)transforming a plant cell with an expression vector comprising a CCSRPcoding nucleic acid, and (b) generating from the plant cell a transgenicplant with an increased tolerance to environmental stress as compared toa wild type variety of the plant. In preferred embodiments, the CCSRPand CCSRP coding nucleic acid are as described below.

The present invention further provides a method of identifying a novelCCSRP, comprising (a) raising a specific antibody response to a CCSRP,or fragment thereof, as described below; (b) screening putative CCSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel CCSRP; and(c) identifying from the bound material a novel CCSRP in comparison toknown CCSRP. Alternatively, hybridization with nucleic acid probes asdescribed below can be used to identify novel CCSRP nucleic acids.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a CCSRP inthe plant, wherein the CCSRP is as described below. The inventionprovides that this method can be performed such that the stresstolerance is either increased or decreased. Preferably, stress toleranceis increased in a plant via increasing expression of a CCSRP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the plant expression vector ppBPSsc022containing the super promoter driving the expression of SEQ ID NO: 4, 5,or 6 (“Desired Gene”). The components are: NPTII kanamycin resistancegene, AtAct2-i promoter, OCS3 terminator, NOSpA terminator (Jefferson etal., 1987 EMBO J 6:3901-7).

FIG. 2 shows the results of a drought stress test with over-expressingPpCC-1 transgenic plants and wild-type Arabidopsis lines. The transgeniclines display a tolerant phenotype. Individual transformant lines areshown.

FIG. 3 shows the results of a freezing stress test with over-expressingPpCC-1 transgenic plants and wild-type Arabidopsis lines. The transgeniclines display a tolerant phenotype. Individual transformant lines areshown.

FIG. 4 shows the results of a drought stress test with over-expressingPpCC-2 transgenic plants and wild-type Arabidopsis lines. The transgeniclines display a tolerant phenotype. Individual transformant lines areshown.

FIG. 5 shows the results of a drought stress test with over-expressingPpCC-3 transgenic plants and wild-type Arabidopsis lines. The transgeniclines display a tolerant phenotype. Individual transformant lines areshown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. In particular, thedesignation of the amino acid sequences as protein “Cell CycleStress-Related Proteins” (CCSRPs), in no way limits the functionality ofthose sequences.

The present invention provides a transgenic plant cell transfonned by aCCSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcell. The invention further provides transgenic plant parts andtransgenic plants containing the plant cells described herein. Alsoprovided is a plant seed produced by a transgenic plant transformed by aCCSRP coding nucleic acid, wherein the seed contains the CCSRP codingnucleic acid, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant. The invention further provides a seed produced by atransgenic plant expressing a CCSRP, wherein the seed contains theCCSRP, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention also provides an agricultural product produced by any ofthe below-described transgenic plants, plant parts and plant seeds.

As used herein, the term “variety” refers to a group of plants within aspecies that share constant characters that separate them from thetypical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

The present invention describes for the first time that thePhyscomitrella patens CCSRPs, CC-1, CC-2 and CC-3, are useful forincreasing a plant's tolerance to environmental stress. Accordingly, thepresent invention provides isolated CCSRPs selected from the groupconsisting of CC-1, CC-2 and CC-3, and homologs thereof. In preferredembodiments, the CCSRP is selected from 1) a Cell Cycle-1 (CC-1) proteinas defined in SEQ ID NO:7; 2) a Cell Cycle-2 (CC-2) protein as definedin SEQ ID NO:8; and 3) a Cell Cycle-3 (CC-3) protein as defined in SEQID NO:9 and homologs and orthologs thereof. Homologs and orthologs ofthe amino acid sequences are defined below.

The CCSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedbelow), the expression vector is introduced into a host cell (asdescribed below) and the CCSRP is expressed in the host cell. The CCSRPcan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a CCSRP polypeptide, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native CCSRP can be isolated from cells (e.g., Physcomitrellapatens), for example using an anti-CCSRP antibody, which can be producedby standard techniques utilizing a CCSRP or fragment thereof.

The invention further provides an isolated CCSRP coding nucleic acid.The present invention includes CCSRP coding nucleic acids that encodeCCSRPs as described herein. In preferred embodiments, the CCSRP codingnucleic acid is selected from 1) a Cell Cycle-1 (CC-1) nucleic acid asdefined in SEQ ID NO:4; 2) a Cell Cycle-2 (CC-2) nucleic acid as definedin SEQ ID NO:5; and 3) a Cell Cycle-3 (CC-3) nucleic acid as defined inSEQ ID NO:6 and homologs and orthologs thereof. Homologs and orthologsof the nucleotide sequences are defined below. In one preferredembodiment, the nucleic acid and protein are isolated from the plantgenus Physcomitrella. In another preferred embodiment, the nucleic acidand protein are from a Physcomitrella patens (P. patens) plant.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, or temperature, or combinations thereof, and inparticular, can be high salinity, low water content or low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of some of the sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated CCSRP nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived (e.g., a Physcomitrella patens cell). Moreover, an“isolated”nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a P. patens CCSRP cDNA can be isolated from a P. patens libraryusing all or portion of one of the sequences of SEQ ID NO:1, SEQ ID NO:2and SEQ ID NO:3. Moreover, a nucleic acid molecule encompassing all or aportion of one of the sequences of SEQ ID NO: 1, SEQ ID NO:2 and SEQ IDNO:3 can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence. For example,mRNA can be isolated from plant cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in SEQ IDNO: 1, SEQ ID NO:2 and SEQ ID NO:3. A nucleic acid molecule of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a CCSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6. These cDNAs comprise sequencesencoding the CCSRPs (i.e., the “coding region”, indicated in Table 1),as well as 5′ untranslated sequences and 3′ untranslated sequences. Itis to be understood that SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6comprise both coding regions and 5′ and 3′ untranslated regions.Alternatively, the nucleic acid molecules of the present invention cancomprise only the coding region of any of the sequences in SEQ ID NO:4,SEQ ID NO:5 or SEQ ID NO:6 or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as L an “ORF position”. The present invention also includesCCSRP coding nucleic acids that encode CCSRPs as described herein.Preferred is a CCSRP coding nucleic acid that encodes a CCSRP selectedfrom the group consisting of, CC-1 (SEQ ID NO:7), CC-2 (SEQ ID NO:8) andCC-3 (SEQ ID NO:9).

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO:4, SEQID NO:5 and SEQ ID NO:6, for example, a fragment which can be used as aprobe or primer or a fragment encoding a biologically active portion ofa CCSRP. The nucleotide sequences determined from the cloning of theCCSRP genes from P. patens allow for the generation of probes andprimers designed for use in identifying and/or cloning CCSRP homologs inother cell types and organisms, as well as CCSRP homologs from othermosses and related species.

Portions of proteins encoded by the CCSRP nucleic acid molecules of theinvention are preferably biologically active portions of one of theCCSRPs described herein. As used herein, the term “biologically activeportion of” a CCSRP is intended to include a portion, e.g., adomain/motif, of a CCSRP that participates in a stress toleranceresponse in a plant, has an activity as set forth in Table 1, orparticipates in the transcription of a protein involved in a stresstolerance response in a plant. To determine whether a CCSRP, or abiologically active portion thereof, can participate in transcription ofa protein involved in a stress tolerance response in a plant, a stressanalysis of a plant comprising the CCSRP may be performed. Such analysismethods are well known to those skilled in the art, as detailed inExample 7. More specifically, nucleic acid fragments encodingbiologically active portions of a CCSRP can be prepared by isolating aportion of one of the sequences in SEQ ID NO:7, SEQ ID NO:8 and SEQ IDNO:9, expressing the encoded portion of the CCSRP or peptide (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the CCSRP or peptide.

Biologically active portions of a CCSRP are encompassed by the presentinvention and include peptides comprising amino acid sequences derivedfrom the amino acid sequence of a CCSRP, e.g., an amino acid sequence ofSEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, or the amino acid sequence ofa protein homologous to a CCSRP, which include fewer amino acids than afull length CCSRP or the full length protein which is homologous to aCCSRP, and exhibit at least one activity of a CCSRP. Typically,biologically active portions (e.g., peptides which are, for example, 5,10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids inlength) comprise a domain or motif with at least one activity of aCCSRP. Moreover, other biologically active portions in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the activities describedherein. Preferably, the biologically active portions of a CCSRP includeone or more selected domains/motifs or portions thereof havingbiological activity.

The invention also provides CCSRP chimeric or fusion proteins. As usedherein, a CCSRP “chimeric protein” or “fusion protein” comprises a CCSRPpolypeptide operatively linked to a non-CCSRP polypeptide. A CCSRPpolypeptide refers to a polypeptide having an amino acid sequencecorresponding to a CCSRP, whereas a non-CCSRP polypeptide refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the CCSRP, e.g., a protein thatis different from the CCSRP and is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the CCSRP polypeptide and the non-CCSRPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-CCSRPpolypeptide can be fused to the N-terminus or C-terminus of the CCSRPpolypeptide. For example, in one embodiment, the fusion protein is aGST-CCSRP fusion protein in which the CCSRP sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant CCSRPs. In another embodiment, the fusionprotein is a CCSRP containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a CCSRP can be increased through use of aheterologous signal sequence.

Preferably, a CCSRP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A CCSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the CCSRP.

In addition to fragments and fusion proteins of the CCSRPs describedherein, the present invention includes homologs and analogs of naturallyoccurring CCSRPs and CCSRP encoding nucleic acids in a plant. “Homologs”are defined herein as two nucleic acids or proteins that have similar,or “homologous”, nucleotide or amino acid sequences, respectively.Homologs include allelic variants, orthologs, paralogs, agonists andantagonists of CCSRPs as defined hereafter. The term “homolog” furtherencompasses nucleic acid molecules that differ from one of thenucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6(and portions thereof) due to degeneracy of the genetic code and thusencode the same CCSRP as that encoded by the nucleotide sequences shownin SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 . As used herein a “naturallyoccurring” CCSRP refers to a CCSRP amino acid sequence that occurs innature. Preferably, a naturally occurring CCSRP comprises an amino acidsequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8and SEQ ID NO:9.

An agonist of the CCSRP can retain substantially the same, or a subset,of the biological activities of the CCSRP. An antagonist of the CCSRPcan inhibit one or more of the activities of the naturally occurringform of the CCSRP. For example, the CCSRP antagonist can competitivelybind to a downstream or upstream member of the cell membrane componentmetabolic cascade that includes the CCSRP, or bind to a CCSRP thatmediates transport of compounds across such membranes, therebypreventing translocation from taking place.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs and paralogs of a CCSRP cDNA can be isolated based ontheir identity to the Physcomitrella patens CCSRP nucleic acidsdescribed herein using CCSRP cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. In an alternative embodiment,homologs of the CCSRP can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the CCSRP for CCSRPagonist or antagonist activity. In one embodiment, a variegated libraryof CCSRP variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of CCSRP variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential CCSRP sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofCCSRP sequences therein. There are a variety of methods that can be usedto produce libraries of potential CCSRP homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene is then ligated into an appropriate expression vector.Use of a degenerate set of genes allows for the provision, in onemixture, of all of the sequences encoding the desired set of potentialCCSRP sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A., 1983 Tetrahedron 39:3;Itakura et al., 1984 Annu. Rev. Biochem. 53:323; Itakura et al., 1984Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the CCSRP coding regions can beused to generate a variegated population of CCSRP fragments forscreening and subsequent selection of homologs of a CCSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a CCSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the CCSRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CCSRP homologs. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify CCSRP homologs (Arkin and Yourvan, 1992 PNAS 89:7811-7815;Delgrave et al., 1993 Protein Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedCCSRP library, using methods well known in the art. The presentinvention further provides a method of identifying a novel CCSRP,comprising (a) raising a specific antibody response to a CCSRP, or afragment thereof, as described above; (b) screening putative CCSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel CCSRP; and(c) analyzing the bound material in comparison to known CCSRP, todetermine its novelty.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 and amutant form thereof), the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of one protein ornucleic acid for optimal alignment with the other protein or nucleicacid). The amino acid residues at corresponding amino acid positions ornucleotide positions are then compared. When a position in one sequence(e.g., one of the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9)is occupied by the same amino acid residue at the corresponding positionin the other sequence (e.g., a mutant form of the sequence selected fromthe polypeptide of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9), then themolecules are homologous at that position (i.e., as used herein aminoacid or nucleic acid “homology” is equivalent to amino acid or nucleicacid “identity”). The same type of comparison can be made between twonucleic acid sequences.

The percent homology between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %homology=numbers of identical positions/total numbers of positions×100).Preferably, the amino acid sequences included in the present inventionare at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80%, 80-90%, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In yetanother embodiment, at least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence encoded by a nucleic acid sequence shownin SEQ ID NO:4,SEQ ID NO:5 or SEQ ID NO:6 . In other embodiments, thepreferable length of sequence comparison for proteins is at least 15amino acid residues, more preferably at least 25 amino acid residues,and most preferably at least 35 amino acid residues.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown inSEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 , or a portion thereof. Thepreferable length of sequence comparison for nucleic acids is at least75 nucleotides, more preferably at least 100 nucleotides and mostpreferably the entire coding region of the nucleic acid.

It is also preferable that the homologous nucleic acid molecule of theinvention encodes a protein or portion thereof which includes an aminoacid sequence which is sufficiently homologous to an amino acid sequenceof SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 such that the protein orportion thereof maintains the same or a similar function as the aminoacid sequence to which it is compared. Functions of the CCSRP amino acidsequences of the present invention include the ability to participate ina stress tolerance response in a plant, or more particularly, toparticipate in the transcription of a protein involved in a stresstolerance response in a Physcomitrella patens plant. Examples of suchactivities are described in Table 1.

In addition to the above-described methods, a determination of thepercent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403-410).

BLAST nucleic acid searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleic acid sequences homologous tothe CCSRP nucleic acid molecules of the invention. Additionally, BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to CCSRPs of thepresent invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997Nucleic Acids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used toobtain amino acid sequences homologous to the CCSRPs of the presentinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997 NucleicAcids Res. 25:3389-3402). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. Another preferred non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe algorithm of Myers and Miller (CABIOS 1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) that is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4 can be used.

Finally, homology between nucleic acid sequences can also be determinedusing hybridization techniques known to those of skill in the art.Accordingly, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to one of the nucleotide sequences shown in SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6 , or a portion thereof. Moreparticularly, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:4, SEQ ID NO:5 or SEQ ID NO:6 . In other embodiments, the nucleicacid is at least 30, 50, 100, 250 or more nucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, 6.3.1-6.3.6, John Wiley& Sons, N.Y. (1989). A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 corresponds to anaturally occurring nucleic acid molecule. As used herein, a “naturallyoccurring” nucleic acid molecule refers to an RNA or DNA molecule havinga nucleotide sequence that occurs in nature (e.g., encodes a naturalprotein). In one embodiment, the nucleic acid encodes a naturallyoccurring Physcomitrella patens CCSRP.

Using the above-described methods, and others known to those of skill inthe art, one of ordinary skill in the art can isolate homologs of theCCSRPs comprising amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8or SEQ ID NO:9. One subset of these homologs is allelic variants. Asused herein, the term “allelic variant” refers to a nucleotide sequencecontaining polymorphisms that lead to changes in the amino acidsequences of a CCSRP and that exist within a natural population (e.g., aplant species or variety). Such natural allelic variations can typicallyresult in 1-5% variance in a CCSRP nucleic acid. Allelic variants can beidentified by sequencing the nucleic acid sequence of interest in anumber of different plants, which can be readily carried out by usinghybridization probes to identify the same CCSRP genetic locus in thoseplants. Any and all such nucleic acid variations and resulting aminoacid polymorphisms or variations in a CCSRP that are the result ofnatural allelic variation and that do not alter the functional activityof a CCSRP, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding CCSRPs from the same or otherspecies such as CCSRP analogs, orthologs and paralogs, are intended tobe within the scope of the present invention. As used herein, the term“analogs” refers to two nucleic acids that have the same or similarfunction, but that have evolved separately in unrelated organisms. Asused herein, the term “orthologs” refers to two nucleic acids fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode proteins having the same orsimilar functions. As also used herein, the term “paralogs” refers totwo nucleic acids that are related by duplication within a genome.Paralogs usually have different functions, but these functions may berelated (Tatusov, R. L. et al. 1997 Science 278(5338):631-637). Analogs,orthologs and paralogs of a naturally occurring CCSRP can differ fromthe naturally occurring CCSRP by post-translational modifications, byamino acid sequence differences, or by both. Post-translationalmodifications include in vivo and in vitro chemical derivatization ofpolypeptides, e.g., acetylation, carboxylation, phosphorylation, orglycosylation, and such modifications may occur during polypeptidesynthesis or processing or following treatment with isolated modifyingenzymes. In particular, orthologs of the invention will generallyexhibit at least 80-85%, more preferably 90%, and most preferably 95%,96%, 97%, 98% or even 99% identity or homology with all or part of anaturally occurring CCSRP amino acid sequence and will exhibit afunction similar to a CCSRP. Orthologs of the present invention are alsopreferably capable of participating in the stress response in plants. Inone embodiment, the CCSRP orthologs maintain the ability to participatein the metabolism of compounds necessary for the construction ofcellular membranes in Physcomitrella patens, or in the transport ofmolecules across these membranes.

In addition to naturally-occurring variants of a CCSRP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 , thereby leading to changes inthe amino acid sequence of the encoded CCSRP, without altering thefunctional ability of the CCSRP. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in a sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ IDNO:6 . A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of one of the CCSRPs withoutaltering the activity of said CCSRP, whereas an “essential” amino acidresidue is required for CCSRP activity. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conserved inthe domain having CCSRP activity) may not be essential for activity andthus are likely to be amenable to alteration without altering CCSRPactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CCSRPs that contain changes in amino acid residuesthat are not essential for CCSRP activity. Such CCSRPs differ in aminoacid sequence from a sequence contained in SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9, yet retain at least one of the CCSRP activities describedherein. In one embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence encoding a protein, wherein the protein comprisesan amino acid sequence at least about 50% homologous to an amino acidsequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. Preferably, theprotein encoded by the nucleic acid molecule is at least about 50-60%homologous to one of the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQID NO:9, more preferably at least about 60-70% homologous to one of thesequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, even morepreferably at least about 70-80%, 80-90%, 90-95% homologous to one ofthe sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, and mostpreferably at least about 96%, 97%, 98%, or 99% homologous to one of thesequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. The preferredCCSRP homologs of the present invention are preferably capable ofparticipating in the a stress tolerance response in a plant, or moreparticularly, participating in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant, or haveone or more activities set forth in Table 1.

An isolated nucleic acid molecule encoding a CCSRP homologous to aprotein sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:6 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a CCSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a CCSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a CCSRP activity described herein to identify mutants that retainCCSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6 , the encoded protein can be expressedrecombinantly and the activity of the protein can be determined byanalyzing the stress tolerance of a plant expressing the protein asdescribed in Example 7.

In addition to the nucleic acid molecules encoding the CCSRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire CCSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a CCSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of , , , comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding a CCSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of oneof the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:6 , or a portion thereof. A nucleic acid molecule that iscomplementary to one of the nucleotide sequences shown in SEQ ID NO:4,SEQ ID NO:5 and SEQ ID NO:6 is one which is sufficiently complementaryto one of the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5 andSEQ ID NO:6 such that it can hybridize to one of the nucleotidesequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 , therebyforming a stable duplex.

Given the coding strand sequences encoding the CCSRPs disclosed herein(e.g., the sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:6 ), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof CCSRP mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of CCSRPmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of CCSRP mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CCSRP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987 Nucleic Acids. Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes described in Haselhoff andGerlach, 1988 Nature 334:585-591) can be used to catalytically cleaveCCSRP mRNA transcripts to thereby inhibit translation of CCSRP mRNA. Aribozyme having specificity for a CCSRP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a CCSRP cDNA, asdisclosed herein (i.e., SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 ) or onthe basis of a heterologous sequence to be isolated according to methodstaught in this invention. For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina CCSRP-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CCSRP mRNA canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel, D. andSzostak, J.W., 1993 Science 261:1411-1418.

Alternatively, CCSRP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a CCSRPnucleotide sequence (e.g., a CCSRP promoter and/or enhancer) to formtriple helical structures that prevent transcription of a CCSRP gene intarget cells. See generally, Helene, C., 1991 Anticancer Drug Des.6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J., 1992 Bioassays 14(12):807-15.

In addition to the CCSRP nucleic acids and proteins described above, thepresent invention encompasses these nucleic acids and proteins attachedto a moiety. These moieties include, but are not limited to, detectionmoieties, hybridization moieties, purification moieties, deliverymoieties, reaction moieties, binding moieties, and the like. A typicalgroup of nucleic acids attached to a moiety are probes and primers.Probes and primers typically comprise a substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 , an anti-sensesequence of one of the sequences set forth in SEQ ID NO:4, SEQ ID NO:5and SEQ ID NO:6 , or naturally occurring mutants thereof. Primers basedon a nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 canbe used in PCR reactions to clone CCSRP homologs. Probes based on theCCSRP nucleotide sequences can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of agenomic marker test kit for identifying cells which express a CCSRP,such as by measuring a level of a CCSRP-encoding nucleic acid, in asample of cells, e.g., detecting CCSRP mRNA levels or determiningwhether a genomic CCSRP gene has been mutated or deleted.

In particular, a useful method to ascertain the level of transcriptionof the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: N.Y.). This information at least partiallydemonstrates the degree of transcription of the transformed gene. Totalcellular RNA can be prepared from cells, tissues or organs by severalmethods, all well-known in the art, such as that described in Bormann,E. R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence orrelative quantity of protein translated from this MRNA, standardtechniques, such as a Western blot, may be employed. These techniquesare well known to one of ordinary skill in the art. (See, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork).

The invention further provides an isolated recombinant expression vectorcomprising a CCSRP nucleic acid as described above, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., CCSRPs, mutant forms of CCSRPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of CCSRPs in prokaryotic or eukaryotic cells. For example,CCSRP genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (see Romanos, M. A. et al., 1992 Foreign gene expression inyeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al.,1991 Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P. J.,1991 Gene transfer systems and vector development for filamentous fungi,in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p.1-28, Cambridge University Press: Cambridge), algae (Falciatore et al.,1999 Marine Biotechnology 1(3):239-251), ciliates of the types:Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in PCT Application No. WO 98/01572 and multicellularplant cells (see Schmidt, R. and Willmitzer, L., 1988 High efficiencyAgrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana leaf and cotyledon explants, Plant Cell Rep. 583-586); PlantMolecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter6/7, S.71-119 (1993); F. F. White, B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,eds. Kung und R. Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu.Rev. Plant Physiol. Plant Molec. Biol. 42:205-225 and references citedtherein) or mammalian cells. Suitable host cells are discussed furtherin Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press: San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of a recombinantprotein; 2) to increase the solubility of a recombinant protein; and 3)to aid in the purification of a recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the CCSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant CCSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

In another embodiment, the CCSRP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., 1987 EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987 Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel, C.A.M.J.J. & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28,Cambridge University Press: Cambridge.

Alternatively, the CCSRPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

In yet another embodiment, a CCSRP nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2^(nd) , ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987 Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988 Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989 EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983 Cell 33:729-740; Queen andBaltimore, 1983 Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989 PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985 Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss, 1990 Science 249:374-379)and the fetoprotein promoter (Campes and Tilghman, 1989 Genes Dev.3:537-546).

In another embodiment, the CCSRPs of the invention may be expressed inunicellular plant cells (such as algae) (see Falciatore et al., 1999Marine Biotechnology 1(3):239-251 and references therein) and plantcells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plant cells and operably linked sothat each sequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tuniefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable.

As plant gene expression is very often not limited on transcriptionallevels, a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene product in itsappropriate cell compartment (for review see Kermode, 1996 Crit. Rev.Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner. Examplesof such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992 Plant J. 2:397-404) and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814) orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al. (1993 Mol. Gen.Genet. 236:331-340).

Especially preferred are those promoters that confer gene expression inspecific tissues and organs, such as guard cells and the root haircells. Suitable promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al., 1991 Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoterfrom Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoterfrom Brassica (PCT Application No. WO 91/13980) or the legumin B4promoter (LeB4; Baeumlein et al., 1992 Plant Journal, 2(2):233-9) aswell as promoters conferring seed specific expression in monocot plantslike maize, barley, wheat, rye, rice, etc. Suitable promoters to noteare the 1pt2 or 1pt1-gene promoter from barley (PCT Application No. WO95/15389 and PCT Application No. WO 95/23230) or those described in PCTApplication No. WO 99/16890 (promoters from the barley hordein-gene,rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadingene, wheat glutelin gene, maize zein gene, oat glutelin gene, Sorghumkasirin-gene and rye secalin gene).

Also especially suited are promoters that confer plastid-specific geneexpression since plastids are the compartment where lipid biosynthesisoccurs. Suitable promoters are the viral RNA-polymerase promoterdescribed in PCT Application No. WO 95/16783 and PCT Application No. WO97/06250 and the clpP-promoter from Arabidopsis described in PCTApplication No. WO 99/46394.

The invention further provides a recombinant expression vectorcomprising a CCSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a CCSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews-Trends in Genetics, Vol. 1(1) 1986 and Mol et al.,1990 FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aCCSRP can be expressed in bacterial cells such as C. glutamicum, insectcells, fungal cells or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms like C. glutamicum. Other suitable host cells are knownto those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratorymanuals such as Methods in Molecular Biology, 1995, Vol. 44,Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa,N.J.. As biotic and abiotic stress tolerance is a general trait wishedto be inherited into a wide variety of plants like maize, wheat, rye,oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed andcanola, manihot, pepper, sunflower and tagetes, solanaceous plants likepotato,

1. A transgenic plant cell transformed with an isolated polynucleotideselected from the group consisting of: a) a polynucleotide having asequence as set forth in SEQ ID NO:6; and b) a polynucleotide encoding apolypeptide having a sequence as set forth in SEQ ID NO:9.
 2. The plantcell of claim 1, wherein the polynucleotide has the sequence as setforth in SEQ ID NO:6.
 3. The plant cell of claim 1, wherein thepolynucleotide encodes the polypeptide having the sequence as set forthin SEQ ID NO:9.
 4. A transgenic plant transformed with an isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:6; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:9.
 5. The plant of claim 4, wherein the polynucleotide has thesequence as set forth in SEQ ID NO:6.
 6. The plant of claim 4, whereinpolynucleotide encodes the polypeptide having the sequence as set forthin SEQ ID NO:9.
 7. The plant of claim 4, wherein the plant is a monocot.8. The plant of claim 4, wherein the plant is a dicot.
 9. The plant ofclaim 4, wherein the plant is selected from the group consisting ofmaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grasses, and a foragecrop plant.
 10. A seed which is true breeding for a transgene comprisinga polynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:6; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:9.
 11. The seed of claim 10, wherein the polynucleotide hasthe sequence as set forth in SEQ ID NO:6.
 12. The seed of claim 10,wherein the polynucleotide encodes the polypeptide having the sequenceas set forth in SEQ ID NO:9.
 13. An isolated nucleic acid comprising apolynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:6; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:9.
 14. The isolated nucleic acid of claim 13, wherein thepolynucleotide has the sequence as set forth in SEQ ID NO:6.
 15. Theisolated nucleic acid of claim 13, wherein the polynucleotide encodesthe polypeptide having the sequence as set forth in SEQ ID NO:9.
 16. Amethod of producing a drought-tolerant transgenic plant, the methodcomprising the steps of: a) transforming a plant cell with an expressionvector comprising a polynucleotide encoding a polypeptide having asequence as set forth in SEQ ID NO:9; b) growing the transformed plantcell to generate transgenic plants; and c) screening the transgenicplants generated in step b) to identify a transgenic plant thatexspresses the polypeptide and exhibits increased tolerance to droughtstress as compared to a wild type variety of the plant.
 17. The methodof claim 16, wherein the polynucleotide has a sequence as set forth inSEQ ID NO: 6.